PROJECT SUMMARY Chronic exposure to nicotine in tobacco products results in numerous health consequences (lung cancer, emphysema, hypertension, etc.) and accounts for over 6 million deaths per year. Relapse rates are high among those who attempt to quit smoking, and pharmacotherapies that seek to foster smoking cessation have moderate effectiveness. Thus, there is a significant unmet need for more effective strategies to treat nicotine dependence. Development of such strategies requires a more detailed understanding of the biological mechanisms leading to nicotine addiction. An essential goal related to mechanistic studies on nAChRs is gaining a better understanding of the location and activity of nAChRs in discrete sites within individual nerve cells. It is also critical that we connect this location/activity information to the various neurochemically-defined (e.g. dopamine, GABA, glutamate) cell types within the brain reward pathways. For these specific cell types in the reward pathway, recent research points to a highly complex input/output relationship. We identified nAChR expression in glutamate-producing neurons in the ventral tegmental area (VTA), and have demonstrated that these receptors enable nicotine- or ACh-mediated alterations in synaptic transmission within the VTA. However, several gaps in knowledge remain, which we will address in this project. First (in Aim 1), we will identify the location within VTA glutamate neurons where nAChRs show the greatest functional activity. This will be done using electrophysiological recordings during 2-photon imaging of neurons in brain slices. This approach will be coupled with studies using a novel photoactivatable nicotine, which we recently introduced. Directly connecting this structural and functional information is critical to fully understanding how nAChRs modulate glutamate transmission in the VTA. In Aim 2, we will answer questions related to nAChR modulation of cell-cell communication within the VTA. Glutamate neurons in this nucleus directly impinge on local DA/GABA neurons, activity which is modulated by nAChR activity on glutamate cells. We will investigate the mechanisms underlying this synaptic communication using optogenetics, photostimulation techniques, and 2-photon microscopy. In Aim 3, we move our queries to the behaving animal to determine which of the components investigated in Aims 1-2 are most important during animal behavior. Using fiber photometry, we will image Ca2+ activity in VTA neurons during acute nicotine exposure and during acquisition/expression of nicotine conditioned reward behavior. To complement these experiments, we will also use chemogenetics to determine whether VTA glutamate neurons are important for nicotine reward-like behavior. By determining how cholinergic mechanisms map onto the complexity (neurochemical and connectivity) of cell types in the VTA, this project will significantly advance our understanding of cholinergic neurotransmission. These studies could also lead to novel treatments for addiction or novel hypotheses about reward system function/activity.